The present invention relates to the use of NMR spectroscopy and biological profiling, in combination with computer-based statistical procedures, in the standardisation and quality control of medicinal plant materials.
Many societies around the world have developed, through the centuries, a system of traditional medicine relying largely on the use of plants and herbs as therapeutic substances. As used herein, the term xe2x80x9cplantxe2x80x9d encompasses plants, including herbs, and fungi.
In recent years there has been a significant growth of interest amongst the general public in the direct use of plants and plant extracts as health modifying agents, for instance Panax ginseng, Allium sativum (garlic), Ginkgo biloba, Hypericum perforatum (St John""s wort), Echinacea angustifolia and Aloe vera. These are currently available on the market as herbal products and dietary supplements and annual sales of these products worldwide currently run to tens of billions of dollars. In spite of this marketing potential the mainstream pharmaceutical industry has not so far directed its attention to the development of medicinal products derived from plants. This is due in part to problems associated with the complex nature and inherent non-uniformity of plant materials, including the lack of an established system by which drug regulatory approval for such products can be secured.
The materials used in herbal and plant based medicine are usually whole plants, parts of plants or extracts of plants or fungi. Since plant and fungal materials contain many different chemical components the materials are, by definition, complex mixtures. This makes it very difficult to standardize and control the quality of the materials. Many of the remedies employed in traditional Chinese medicine and Ayurvedic medicine mentioned above are mixtures of two or more plant-based components. They are therefore effectively mixtures of mixtures and thus even more difficult to analyse than herbal remedies based on a single plant material. Furthermore, the recipes and methods of manufacture used for such remedies frequently remain undisclosed. These factors make it very difficult to ensure that two samples of a given remedy, obtained from disparate sources and ostensibly identical, do in fact contain the same mixture of ingredients. This problem, which leads to difficulties in controlling the quality of such materials, has so far limited the acceptability of Eastern herbal remedies to Western herbal practitioners.
The plants used in the practice of herbal medicine are frequently unavailable locally and therefore need to be obtained from sources which are remote from the end user. However, the supply of such plants from remote locations can be erratic and inaccurate, particularly because no detailed monographs including identity and quality standards exist for many such plants. The complex mixture of ingredients found in medicinal plants will in any event vary widely in type and concentration depending on many factors including the botanical origin, the location where the plant is grown, the time of year when the plant is harvested, the conditions under which the material is stored and processed and the extraction procedure used. When these plants are in turn mixed with other plants, for instance according to traditional Chinese herbal recipes, there is considerable scope for variability in the resulting product.
It is virtually impossible at present to provide any assurance that samples of a given plant material obtained from disparate sources will possess a uniform identity and biological activity. The present invention addresses this problem by providing a means of standardising a medicinal plant material. The approach takes account of the totality of the components of the plant material without demanding any inquiry into the intrinsic nature of either the components themselves or the plant""s biochemistry. It involves interrogating the consistency of a plant material on both a chemical level and a biological level.
Thus the process of the present invention provides a means of defining a standard for a given medicinal plant material on the basis of a known sample of the material which possesses the particular property desired for the standard. A specification for the standard is established by submitting the known sample to (a) a combination of NMR spectroscopy and a computer-based pattern recognition technique and (b) one or more biological profiling techniques, and defining the results thus obtained as the standard specification. Subsequent xe2x80x9ccandidatexe2x80x9d samples of the said plant material can then be tested for compliance with the standard. They can be accepted or rejected depending on whether they give analytical results which fall within or outside either part or all of the established specification.
The present invention accordingly provides a process for establishing a standard specification for a medicinal plant material, the process comprising:
(i) preparing a test solution or test extract of a sample of the medicinal plant material which is known to possess the or each property desired for the standard;
(ii) submitting the said solution or extract to two or more analytical methods including (a) a combination of NMR spectroscopy and a computer-based pattern recognition technique, and (b) one or more biological profiling techniques;
(iii) obtaining results from the analytical methods used in step (ii); and
(iv) defining a standard specification for the said plant material on the basis of the results obtained in step (iii).
The standard specification resulting from step (iv) is thus based on the results of NMR spectroscopy and computer-based pattern recognition as well as on the results of one or more biological profiling techniques. However, when candidate samples of the medicinal plant material are tested for compliance with the standard they need not all be submitted to both the NMR spectroscopic analysis and the biological profiling. Rather, all candidate samples are submitted to NMR spectroscopy and pattern recognition whilst only selected candidate samples, for instance taken periodically from batches of the medicinal plant material, are tested for compliance with the biological profiling aspect of the standard specification. The purpose of this is to rely principally on the analytical method which is best suited to convenient and fast operation on a high-throughput scale. This is the NMR spectroscopic and pattern recognition analysis. The biological techniques are typically used on a random basis for validation and reinforcement of the decisions being made to accept or reject the candidate samples on the basis of the NMR spectroscopic and pattern recognition results.
Accordingly the invention further provides a process for providing a sample of a medicinal plant material, which sample complies with a previously defined standard specification for that plant material, the process comprising:
(ixe2x80x2) preparing a test solution or test extract of a candidate sample of the medicinal plant material;
(iixe2x80x2) submitting the said solution or extract to analysis by a combination of NMR spectroscopy and a computer-based pattern recognition technique;
(iiixe2x80x2) obtaining results from the analysis of step (iixe2x80x2); and
(ivxe2x80x2) selecting the candidate sample if the results obtained in step (iiixe2x80x2) comply with the standard specification for the said material established in step (iv) of the process above.
This process is conveniently carried out on a high-throughput batch scale. Candidate samples are taken from batches of the same plant material and submitted to steps (ixe2x80x2) to (ivxe2x80x2). When the candidate sample is selected for submission to biological profiling in addition to the NMR spectroscopic and pattern recognition analysis the above step (ivxe2x80x2) is replaced by the following steps:
(ivxe2x80x2a) submitting the solution or extract prepared in step (ixe2x80x2) to one or more biological profiling techniques;
(ivxe2x80x2b) obtaining results from the or each technique used in step (ivxe2x80x2a); and
(ivxe2x80x2c) selecting the candidate sample if the results obtained in steps (iiixe2x80x2) and (ivxe2x80x2b) comply with the standard specification for the said material as established in step (iv) of the process above.
The biological profiling techniques are discussed in more detail later on.
The xe2x80x9cproperty desired for the standardxe2x80x9d in the context of the present invention may be any property or quality possessed by, or attributed to, a medicinal plant material. Examples of this include a clinically proven therapeutic efficacy, a pharmaceutical grade quality, a particular variety of a given plant genus, an authenticated origin (in terms of either growth location or commercial batch) and a particular pathological state. The pathological state in question may be a given level of maturity, dictated for instance by the time of harvesting, or an established resistance to a parasite, herbicide, insecticide or other agent with potential for damage to the plant in question.
In a preferred aspect of the invention the sample of medicinal plant material which is xe2x80x9cknown to possess the or each property desired for the standardxe2x80x9d is a sample of authenticated and audited plant material of which the provenance is known. A standard specification is established by submitting that sample to NMR spectroscopy/pattern recognition and biological profiling as described above. Subsequent samples of the same plant material, the origin or quality of which is not known or is in doubt, can then be tested for compliance with the standard specification thus established for the authenticated and audited material.
Nuclear magnetic resonance spectroscopy (NMR) is known by itself as an analytical tool in the investigation of plant materials. One example of its application is in the verification of the authenticity of drinks derived from fruit. In one approach hydrogen-2 NMR spectroscopy has been employed with the technique of site-specific natural isotope fractionation (SNIF-NMR) as a means of establishing the authenticity of fruit juices. For instance, in JAOAC Int. 1996 July-August, 79(4): 917-928 Martin et al describe the use of hydrogen-2 NMR spectroscopy (SNIF-NMR method) to detect fruit juices which have been adulterated with added beet sugar. The technique relies on the fact that, when a fruit juice or fruit concentrate is fermented, the proportion of the resulting ethanol molecules which are mono-deuterated on the methyl site decreases with the addition of beet sugar. Thus any fruit juice sample to which beet sugar has been added will have a significantly lower (D/H) isotope ratio than a corresponding authentic sample. This technique has also been applied to the detection of wine chaptalisation using hydrogen-2 NMR spectroscopy, as reported for example in J.Chim. Phys.-Chim Biol. 1983, vol 80, pp 293-297 by Martin et al.
Hydrogen-1 NMR spectroscopy cannot itself conveniently be applied to plant materials because it generates spectra that are too complicated to be analysed visually. A solution to this problem, reported for instance by Kowalski and Bender in J. Am. Chem. Soc. 1972, 94, 5632-5639, is to analyse the data by appropriate multivariate statistical analysis, for example principal component analysis (PCA). This is a technique of pattern recognition where the dimensionality of the data is reduced by combining correlated variables (peaks in the spectrum) to form a new smaller set of independent orthogonal variables called principal components (PCs). These PCs are ordered according to their ability to explain the variance contained in the original data. A projection of the samples into a space spanned by the first PCs provides an insight into the similarity or dissimilarity of the samples with respect to their biochemical composition. Unknown or test samples can also be projected onto this space and can thus often visually be compared with the reference samples (Vogels et al, J. Agric. Food Chem. 44,175-180, 1996).
The combination of hydrogen-1 NMR spectroscopy with pattern recognition techniques has been applied as a screening tool in determining the authenticity of orange juice (Vogels et al, 1996 loc. cit.). The adulteration of suspect samples could be detected by this means. The identity of the responsible contaminants was then determined by correlation of the PCA results with particular resonances present in the original NMR spectrum.
A combination of hydrogen-1 NMR spectroscopy and carbon-13 NMR spectroscopy with PCA has also been used to differentiate wines on the basis of their origin (Vogels et al, Chemometrics and Intelligent Laboratory Systems: Laboratory Information Management, 21 (1993) 249-258). Discriminant plots of samples originating from different wine-producing regions in Germany showed clustering of the samples by origin in the discriminant space after a supervised method of statistical analysis. Subsequently, reconstructed spectra were prepared from the PCA data to reveal the NMR spectroscopic peaks of the particular wine constituents (for instance monosaccharides such as glucose, mannose, rhamnose and galactose) responsible for the differentiation. Similar studies are reported elsewhere, for instance by Vogels et al in Trends in Flavour Research, Maarse and Van de Heij (Eds), Elsevier, Amsterdam (1994) pp 99-106.
Another application of hydrogen-1 NMR spectroscopy and principal component analysis is reported by Trevisan et al in Chapter 8 of xe2x80x9cCharacterisation of cell suspension cultures of hop, Humulus lupulus L.xe2x80x9d, a thesis presented to the University of Leiden, the Netherlands, pages 95-122, published in 1997. The authors carried out hydrogen-1 NMR spectroscopy and PCA on treated cell extracts with the aim of identifying specific metabolites accumulated by the cells following treatment. They were therefore interested in following specific peaks in the NMR spectrum which were known to be due to individual cell components.
In contrast to these reported methods the NMR spectroscopic and pattern recognition procedure employed in the process of the present invention requires neither an investigation into the biochemistry of the plant being analysed nor a subsequent correlation of the pattern recognition results with particular NMR spectroscopic resonances attributed to specific component(s) of the plant material. Instead it relies upon the information presented by the inherent pattern of clusters derived from NMR data, those data in turn reflecting the totality of the compounds in the plant material which respond to the NMR spectroscopic technique being used.
The NMR spectroscopy combined with computer-based pattern recognition (hereinafter termed NMR spectroscopy/pattern recognition) employed in the process of the invention typically comprises:
(a) submitting the test solution or test extract to NMR spectroscopy and recording one or more NMR spectra; and
(b) submitting the data obtained from the or each NMR spectrum to a multivariate analysis to generate one or more points on a score plot.
A sphere of acceptability is typically defined around the point or points on the score plot generated in step (ii) above when the NMR spectroscopy/pattern recognition analysis is being used to establish a standard specification for a medicinal plant material. That sphere then constitutes part of the specification. Candidate samples of the same material are subsequently accepted or rejected depending whether, when submitted to the NMR spectroscopy/pattern recognition analysis defined above, they give points in step (ii) which fall within or outside the sphere.
The NMR spectroscopy/pattern recognition can be used by itself as a way of standardising samples of medicinal plant materials. Accordingly, in one aspect the present invention provides a process for providing a sample of a medicinal plant material which complies with a previously established standard specification for that material, the process comprising:
(ixe2x80x3) preparing a test solution or test extract of a candidate sample of the said plant material;
(iixe2x80x3) submitting the test solution or test extract to NMR spectroscopy and recording one or more NMR spectra;
(iiixe2x80x3) submitting the data obtained from the or each said NMR spectrum to a multivariate analysis to generate one or more points on a score plot; and
(ivxe2x80x3) selecting the candidate sample as a sample which complies with the said standard specification only if the points generated on the score plot in step (iii) fall within a sphere of acceptability as defined in the standard specification.
The standard specification in this aspect of the invention may be provided by a process which comprises:
(ixe2x80x2xe2x80x3) preparing a test solution or test extract of a sample of the said plant material which is known to possess the or each property desired for the standard;
(iixe2x80x2xe2x80x3) submitting the test solution or test extract to NMR spectroscopy and recording one or more spectra;
(iiixe2x80x2xe2x80x3) submitting the data obtained from the or each said NMR spectrum to a computer-based multi-variate analysis to generate one or more points on a score plot; and
(ivxe2x80x2xe2x80x3) defining a sphere of acceptability around the points generated in step (iiixe2x80x2xe2x80x3) as the, or as part of the, standard specification for the said plant material.
In general multivariate analysis theory, the total data is the product of the scores multiplied by the loadings. The loadings plot can be used to define the contribution of each of the variables (spectral descriptors). A xe2x80x9cscore plotxe2x80x9d is a graphic representation in which samples are projected into the space spanned by two or more principal component axes. Principal component analysis (PCA) is a particular method used to analyse data included in a multivariate analysis. In PCA the position of the samples can be plotted in a score plot in two dimensions where similar samples will tend to form clusters while dissimilar samples will tend to spread over large distances (Kowalski and Bender, 1972, loc. cit. and Trevisan, 1997, loc. cit.).
The context in which the points are generated on the score plot in the process of the present invention must be the same when establishing the standard specification as when analysing candidate samples for compliance with the standard. The components of the methodology used to establish the positioning of the point or points on the score plot for the known sample used to define the standard must be present when the NMR spectroscopic data from the candidate test samples are processed. In practice the data derived from the NMR spectrum of the sample used as the standard are subjected to appropriate manipulation by multivariate statistical methods, for example principal component analysis or canonical variation, together with those of the standard. The sphere of acceptability is defined by limits in the score plot which have been established on the basis of the position in the score plot of points derived from one or more extracts of the known sample. In a preferred aspect of the invention the multivariate analysis is performed using an unsupervised methodology.
The NMR spectroscopic technique used in the invention may involve carrying out hydrogen-1 NMR spectroscopy at high fields in combination with multivariate analysis. In this particular aspect the NMR spectra are typically measured at 400 to 700 MHz. The data derived from them are then analysed by multivariate analysis software, for instance the commercially available xe2x80x9cPirouettexe2x80x9d package.
Examples of the high resolution hydrogen-1 NMR spectroscopic and pattern recognition analysis are discussed by M. L. Anthony et al in Biomarkers 1996, 1, 35-43 and Molecular Pharmacology 46, 199-211, 1994, and by J. O. T. Gibb et al in Comp. Biochem. Physiol. vol. 118B No. 3, pp 587-598, 1997.
As an alternative to 1-dimensional high field hydrogen-1 NMR spectroscopy, 1-dimensional NMR spectroscopy using other NMR-active nuclei such as carbon-13 or hydrogen-2 may be used in the present invention. It is also possible to use a range of 2-dimensional pulse sequence spectroscopic investigations with hydrogen-1 or other NMR-active nuclei such as those mentioned above. The same principles apply in each case, though, and the results are analysed by appropriate multivariate analysis.
The NMR spectra may be normalised or non-normalised before the computer-based pattern recognition is carried out. Normalisation has the effect of removing peak intensity, which is a purely quantitative parameter of the spectra, as a discriminating factor. Normalisation is therefore typically carried out when the main objective of the procedure is to highlight qualitative differences between spectra obtained from different samples. However, in some cases peak intensity may be required as a discriminating factor when absolute quantitative values, for instance potency, are required. In such situations the spectra are non-normalised.
An important advantage of NMR spectroscopy/pattern recognition is that it is not limited by a selective delivery or detection system. Spectra can be recorded without prior purification of the test solution or test extract, thus allowing all components of the sample which possess a proton to contribute to the overall NMR spectrum. Analysis of the spectrum by the multivariate analytical techniques discussed above reveals potential valuable discriminating features of the spectra which can be used with a high degree of precision for the description of the complex mixtures of components contained in plant materials.
It is nonetheless possible in certain cases for the differentiation of samples of plant material on the score plot to be poor, with points deriving from identical samples of a given plant material being spread widely rather than forming a cluster. This loss of similarity arises when there is variation in the NMR spectroscopic shift values of individual components of the plant material. Such variation may be caused, for instance, by the presence of an overwhelmingly high concentration of one particular compound in the plant material or by the modifying effect of pH or metal ions which cause shift values to change. If this problem occurs the test solutions or test extracts of the plant material which are submitted to NMR spectroscopy may be pre-treated to remove the source of error and achieve better clustering in the score plot. In one aspect the process of the invention therefore includes the additional initial step of purifying the test solution or test extract of the candidate sample of plant material prior to submitting it to NMR spectroscopy.
Tea provides a convenient illustration of this principle. Signals due to caffeine dominate the hydrogen-1 NMR spectrum of tea and so when NMR spectroscopic results of tea are processed by multivariate analysis the points on the score plot for samples of the same tea do not form clusters. If caffeine is removed from tea prior to carrying out NMR spectroscopy, for instance by reverse phase chromatography, proper clustering of the samples occurs and the similarities between like samples on the score plot become clear. This is illustrated in Example 2 and accompanying FIGS. 4A and 4B. FIG. 4A is a score plot for untreated tea samples where clustering is indistinct. FIG. 4B is a score plot for pre-treated tea where, in contrast, there is clear clustering which allows positive discrimination.
The NMR spectroscopy/pattern recognition analysis is highly sensitive and has the capacity to differentiate samples of plant material which appear to be identical when analysed by other methodologies. This has again been illustrated using tea. Comparative Example 1 describes the analysis by high performance liquid chromatography (HPLC) of extracts of two different types of tea. The resulting chromatograms are shown in accompanying FIGS. 2 and 3. One experiment used untreated samples (chromatogram A in each of FIGS. 2 and 3) and the other used a treated sample (chromatogram B in each Figure).
The Figures show first of all that HPLC does not clearly distinguish between treated and untreated samples of tea since chromatograms 2A and 2B are virtually identical, as are chromatograms 3A and 3B. Second, the Figures show that HPLC does not have the power to discriminate between different types of tea since the chromatograms of FIG. 2 are virtually identical to those of FIG. 3. In both these respects HPLC contrasts with the NMR spectroscopy/pattern recognition technique used in the present invention as illustrated in Example 2 and the accompanying FIGS. 4A and 4B.
An important application of the process of the present invention is in the standardisation of mixtures of plant materials. Examples of such mixtures include remedies from Traditional Chinese Medicine as discussed above. These are typically mixtures of several different plants and fungi prepared in accordance with recipes that may be many hundreds of years old. To date there has been no analytical technique by which producers of such materials could reliably and consistently differentiate their products from ostensibly identical products sold by competitors under the same name. It has now surprisingly been found that the NMR spectroscopy/pattern recognition technique used in the present invention can provide clear differentiation between samples of a given mixture of plant materials which are supposed to be identical but are obtained from different sources. This is illustrated in Example 5 and accompanying FIG. 7. The process of the invention therefore allows mixtures of plant materials to be differentiated and standardised.
The combination of NMR spectroscopy/pattern recognition and biological profiling is frequently desirable since the complex mixture of compounds in a medicinal plant material may show an overall clinical effect which may derive principally from one particular component but which is considerably modified or potentiated by the presence of other components. Biological profiling can thus provide a quantifiable measure of the biological effects of plants and plant extracts, thereby complementing and/or confirming the information obtained by the NMR spectroscopic and pattern recognition analysis.
The biological profiling techniques used in the process of the invention contribute a means of quality controlling and standardising a desired sample of plant material in terms of its biological activity. It is now recognised that the overall efficacy of plant based medicines does not solely derive from a single active component but is due also to auxiliary compounds which are present in the complex mixture of substances in the plant. The biological profiling techniques used in the process of the invention allow synergistic effects exerted by these auxiliary compounds to be studied without the need to identify the compounds themselves. he synergistic effects which may be observed include, for instance, potentiation of the activity of the principal component, enhancement of the selectivity or bioavailability of the therapeutic substance and suppression of unwanted side effects. This aspect of the biological profiling is particularly useful for substantiating the claim that the use of a whole plant or plant extract in therapy is more beneficial than the use of single components isolated from the plant.
A preferred biological profiling technique is protein analysis, particularly proteomics. This is because changes in protein expression represent the ultimate biological effect irrespective of the particular mechanism of action such as enzyme inhibition, receptor binding inhibition or signal transduction modulation. The term proteome describes the complete set of proteins that is expressed, and modified following expression, by the entire genome in the lifetime of a cell. Proteomics is the study of the proteome using technologies of large-scale protein separation and identification (Nature, vol 402, 1999, p.715). A proteomics analysis is illustrated in Example 6 which follows.
Accordingly, in a preferred aspect of the present invention the biological profiling technique comprises:
(i) providing a target cell selected according to the clinical indication in which the medicinal plant material is active and incubating the target cells with a candidate sample of the said material; and
(ii) subjecting the incubated cells to gel electrophoresis on a 2-D gel and observing the change in protein expression in the cells as a result of exposure to the test sample.
In the process of the invention the above steps (i) and (ii) are carried out on a sample of the plant material which possesses the or each property desired for the standard. The overall pattern of change in protein expression observed in step (ii) is defined as part of the standard specification for that plant material. Candidate samples of the same plant material may then be accepted or rejected depending whether they give the same pattern when submitted to the above steps (i) and (ii).
An appropriate target cell is selected in the first step according to the clinical indication, or disease, which it is desired to model. Following incubation of the cell with the test sample, the proteins in the target cell are separated into individual proteins by two dimensional electrophoresis. Detection and analysis of the resulting protein patterns is typically undertaken using computerized image analysis techniques, and proteins are identified using microsequencing and mass spectroscopy. The results may, if desired, be submitted to computer-based pattern recognition procedures such as principal component analysis, as described above. Changes in protein expression which are detected following incubation of the target cell with the test sample are then compared with the corresponding changes detected following incubation of the target cell with a known sample which has already been shown to possess activity against the clinical indication in question.
The potency of the test sample can be related to the potency of a pharmaceutical grade standard by comparing the concentration of the test sample necessary to produce the response of the standard.
A further biological profiling technique is a receptor binding or enzyme inhibition assay. This can give a quantifiable measure of the biological activity of a plant material. Such an assay may be conducted in accordance with a conventional assay protocol. One example of a suitable assay is a method for screening a plant material as a candidate for the treatment or prophylaxis of cancer or inflammation, the method comprising determining whether the substance suppresses the stimulation of a gene promoter which has been implicated in carcinogenesis or inflammation.
In the process of the invention the plant material typically consists of, or is derived from, a whole plant, a part of a plant, a plant extract or a plant fraction. Preferably the material consists of, or is derived from, one or more of the roots, leaves, buds, flowers, fruit, juice and seeds of a plant.
The inherent variability of plant materials presents a particular problem to the drug regulatory authorities who need to be convinced that a candidate product for pharmaceutical licencing is of a consistent and verifiable quality. The reason for this is that the effectiveness of dosage levels and treatment protocols need to be guaranteed. However, there is no reliable system available at present which allows the identity and activity of a plant based product to be measured against an accepted standard and which is universally applicable to all kinds of plant material. In one aspect, therefore, the invention as defined above represents a solution to that problem and provides a means of establishing a pharmaceutical grade standard for a therapeutic substance which is derived from, or consists of, a plant material. In this aspect the process comprises:
(i) preparing a test solution or test extract of a sample of the said therapeutic substance which is known to be of the desired pharmaceutical grade;
(ii) submitting the test solution or extract to two or more analytical methods including (a) NMR spectroscopy combined with a computer-based pattern recognition technique, and (b) one or more biological profiling techniques;
(iii) obtaining results from the analytical methods in step (ii); and
(iv) defining the pharmaceutical grade standard specification on the basis of the results obtained in step (iii).
The invention further provides a process for producing a pharmaceutical grade therapeutic substance which is derived from, or consists of, a plant material, the process comprising;
(ixe2x80x2) preparing a test solution or test extract of a candidate sample of the therapeutic substance;
(iixe2x80x2) submitting the said solution or extract to analysis by a combination of NMR spectroscopy and a computer-based pattern recognition technique;
(iiixe2x80x2) obtaining results from the analysis of step (iixe2x80x2); and
(ivxe2x80x2) selecting the therapeutic substance as being of pharmaceutical if the results obtained in step (iiixe2x80x2) comply with the specification for the pharmaceutical grade standard established in step (iv) above.
When the candidate sample is selected for submission to biological profiling in addition to the NMR spectroscopic and pattern recognition analysis the above step
(ivxe2x80x2) is replaced by the following steps:
(ivxe2x80x2a) submitting the solution or extract prepared in step (ixe2x80x2) to one or more biological profiling techniques;
(ivxe2x80x2b) obtaining results from the or each technique used in step (ivxe2x80x2a); and
(ivxe2x80x2c) selecting the therapeutic substance as being of pharmaceutical grade if the results obtained in steps (iiixe2x80x2) and (ivxe2x80x2b) comply with the specification for the pharmaceutical grade standard established in step (iv) of the process above.
In these embodiments the NMR spectroscopy, pattern recognition and biological profiling may all be performed as described earlier in the specification.